We propose to (1) develop and test three non-invasive Tools to assess the in vitro viability, structure, and metabolic activity of an engineered tissue in real time and (2) a fourth to assess its viability after grafting in situ in SCID mice. As our model system we will use an engineered tissue, Ex Vivo Produced Oral Mucosal Equivalent (EVPOME), a tissue engineered human oral mucosa developed for intraoral grafting procedures. The EVPOME has been successfully tested in a Phase I human clinical trial and offers a unique engineered tissue model that can be followed during its manufacturing (mfg.) process in vitro. HYPOTHESIS: Non-invasive Tools can be developed and used to assess viability, structure, function and metabolic activity of the cellular component of an engineered tissue, EVPOME, in real time in vitro. Several of these Tools can also be used to assess, in situ, tissue performance of the EVPOME after its engraftment. We will test our hypothesis by developing three non-invasive functional and analytical assays/methods or Tools to assess function, structure/composition and viability of the oral keratinocyte layer(s) of the EVPOME: SPECIFIC AIMS: (1) Test the efficacy of a microfluidic on-line electrophoresis immunoassay on a chip to assay three constitutively secreted proteins of oral keratinocytes important in graft function and survival: human 2-defensin 1 (hBD-1), (an innate antimicrobial), and pro-angiogenic cytokines vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8). (2) Test the efficacy of Quantitative Fluorescence Spectroscopy to examine the metabolic activity of the cellular component of the EVPOME via the redox state of endogenous fluorophores flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). (3) Test the efficacy of Raman Spectroscopy to assess the chemical structure and composition of the EVPOME construct. (4) To assess EVPOME constructs in vitro using Tools developed in Specific Aims 1-3. (5) To assess tissue perfusion and vascularity of the EVPOME, in situ, after grafting into SCID mice, by developing Reflectance Spectroscopy, to be used in conjunction with Fluorescence and Raman Spectroscopy. Our innovative Tools will be used to establish release criteria of an engineered tissue in a more comprehensive fashion, i.e., viability, structure and metabolic activity, compared with presently used assay systems, such as glucose uptake. The development of release criteria in real time will improve the quality control of engineered tissues during the mfg. process and the ability to assess tissue viability in situ, non- invasively, after grafting into animals. Public Health Relevance Statement (provided by applicant): Tissue made outside the body for use in wound repair, burn and other medical therapies needs to go through quality testing before it can be used in human beings. The present day technology either destroys a large part of the manufactured tissue for this testing, or depends on measuring the use of one nutrient, glucose, which the tissue ingests while it is being prepared. We propose to develop, test and use three new methods for checking tissues made outside the body before use in medical therapies. These methods use state of the art technologies to measure metabolism, cell structure and molecules made by the tissue to determine the health of the manufactured tissue before being put into the body. No part of the manufactured tissue will be destroyed when using these new methods. In addition, two of the methods can be used to follow the successful growth of the manufactured tissue after it is placed in the body. We will test the methods in an animal model.